Novobiocin Susceptibility Test

Novobiocin Susceptibility test is usually done to differentiate the coagulase-negative Staphylococci and to identify and isolate as Staphylococcus saprophyticus, which is usually novobiocin resistant.

It is generally performed on urine isolates, especially the urine from women of the reproductive ages, as they contain coagulase negative.

Novobiocin is an aminocoumarin antibiotic, and it is produced from actinomycete Streptomyces nivens, and has its own antibacterial property.

Kloos and Schleifer in the year 1975, detected the simplified scheme for differentiating coagulase-negative Staphylococcus spp.

That includes novobiocin disk test. Staphylococcus saprophyticus, is one of the gram-positive coagulase negative species of Staphylococci.

It is an uropathogenic bacterium which acutes acute infection in the urinary tract.

This infection is uncomplicated, it is often found in young and middle-aged female patients.

Unlike other Coagulative Negative Staphylococcus, Staphylococcus saprophyticus is rarely resistant to the antibiotics and it is active against gram-positive organisms.

S. saprophyticus is one of the promising resistant of the antibiotic novobiocin.

Hence screening for coagulase-negative staphylococci from the urine cultures for novobiocin resistance is reliable presumptive for identifying saprophyticus.

Novobiocin Susceptibility Test Objective

Novobiocin susceptibility test helps to determine the susceptibility pattern of the bacterium with the antibiotic novobiocin.

Novobiocin Susceptibility Test Principle

As mentioned above, novobiocin is an antibiotic which interferes with the unpacking and repacking of DNA during replication of DNA and the bacterial cell cycle.

Novobiocin binds with the DNA gyrase and restricts the activity of adenosine triphosphate.

Susceptibility of the novobiocin is determined by placing a novobiocin antibiotic in a paper disk which is kept on a agar plate and then it is seeded with the organism under investigation.

As organism multiplies during the period of incubation and it produces lawn of confluency growth, cells are exposed to antibiotic diffusing into the agar from the paper disk.

If the bacterial species are susceptible to antibiotic novobiocin, then there will be a formation of transparent zone of inhibition around the area of the disk, which represents the area where the antibiotic concentration prevented the bacterial growth.

If there is no zone of inhibition then, it is said that the organism is resistance to the specific antibiotic used. Usually in the laboratories, Mueller-Hington agar plate is used.

The agar plate is seeded with the test organism to produce a confluent growth on the surface of the agar.

After seeding the test organism is completed, a novobiocin antibiotic disc is applied on the surface of the agar, and incubation is done and further the sensitivity of the organism to the antibiotic determined by performing a Kirby-Bauer method.

Novobiocin Susceptibility Test Plate

Novobiocin disks are prepared by impregnating 5µg of novobiocin into a high quality of about 6mm diameter filter paper disks or it can also be purchased, as it is also commercially available.

Novobiocin Susceptibility Test Procedure

 First the test isolated is kept in the pure culture for about 18 to 72 hours. Suspension is prepared of the test isolate in the tryptic soy broth which is equal to a Mc Farmland 0.5 standard or equivalent.

 Sterile swab is immersed in the suspension and it is rotated against the sides of the tube above the fluid level to remove the excess inoculum.

 Then the expressed swab is collected and inoculated into a blood agar or into the Muller Hinton plate by streaking the swab on the entire agar plate.

 This process is repeated in both the plates.

 After streaking the agar surface is allowed to dry for at least 15 to 20 minutes before applying it on the novobiocin disk.

 Using a sterile swab, lawn of growth is prepared on the entire surface of the plate in three directions and also along the entire edge of the plate.

 Then the forceps are dipped in an alcohol and flamed.

 Then the aseptically flames are used for applying the novobiocin disc to the surface of the inoculated plate.

 The discs are genteelly pressed down using a sterile forceps to ensure whether they adhere to the agar surface.

 The plates are aerobically incubated for about 18 to 24 hours at a temperature of about 35 to 37ºC.

 The diameter of the zone of inhibition is measure using a metric ruler or sliding capillaries.

Novobiocin Susceptibility Test Results

Positive Results: In case, if the results are positive, then the zone of inhibition will be greater than 16mm in diameter and it also indicates that the specific organism is sensitive to the antibiotic.

Negative Results: If the results are negative, then there will be a zone of inhibition which is less than or equal to 16mm, which indicates the novobiocin resistance.

Novobiocin Susceptibility Test Significance

 The CoNS is subdivided into two based on their novobiocin pattern of susceptibility. Here the CoNS group demonstrates the novobiocin susceptibility including epidermis, S. Captitits, S. haemolyticus, S. hominis, S. lugdenensis, S. warneri, S. saccharolyticus and other species.

 The novobiocin resistant group consists of species such as S. saprophyticus, S. xylosus, and kloosii.

 This test is basically used for differentiating Staphylococcus saprophyticus from other Coagulase negative staphylococci, especially in the clinical specimens.